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PubMed1988

Metabolite diffusion into bundle sheath cells from c(4) plants: relation to c(4) photosynthesis and plasmodesmatal function.

The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C(4) plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function.

Journal Article↗
PubMed1990

Coupling of solute transport and cell expansion in pea stems.

As cells expand and are displaced through the elongation zone of the epicotyl of etiolated pea (Pisum sativum L. var Alaska) seedlings, there is little net dilution of the cell sap, implying a coordination between cell expansion and solute uptake from the phloem. Using [14C] sucrose as a phloem tracer (applied to the hypogeous cotyledons), the pattern of label accumulation along the stem closely matched the growth rate pattern: high accumulation in the growing zone, little accumulation in nongrowing regions. Several results suggest that a major portion of phloem contents enters elongating cells through the symplast. We propose that the coordination between phloem transport and cell expansion is accomplished via regulatory pathways affecting both plasmodesmata conductivity and cell expansion.

Biological Transport↗
PubMed1991

The evidence for symplastic Phloem loading.

Experimental protocols used to study the route(s) of phloem loading in source leaves are evaluated; they include the analysis of plasmodesmatal ultrastructure and distribution, mobile compounds in the free space, loading patterns of exogenous sugars, the site of sugar synthesis, and dye coupling. All these methods have drawbacks. There are indications that some plants, especially those that translocate the raffinose series of sugars, load by a symplastic (through plasmodesmata) pathway, but the evidence is weak.

Journal Article↗
PubMed2002

Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus.

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.

DNA, Viral↗
PubMed2003

Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells.

Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized.

Actin Cytoskeleton↗
PubMed1998

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.

Journal Article↗
PubMed2000

A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants.

A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The K(m) for sucrose uptake by AtSUT4 of 11.6 +/- 0.6 mM was approximately 10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.

Amino Acid Sequence↗
PubMed2001

Sucrose export defective1 encodes a novel protein implicated in chloroplast-to-nucleus signaling.

The Sucrose export defective1 (Sxd1) gene of maize was cloned and shown to encode a novel protein conserved between plants and cyanobacteria. The structure of the Sxd1 locus was determined in wild-type plants and two independent sxd1 alleles. Expression analysis demonstrated that the gene was transcribed in all green tissues, with highest levels in maturing leaf blades. In situ hybridization studies revealed high levels of Sxd1 mRNA in bundle sheath cells, with lower levels within the mesophyll. The SXD1 protein was localized to chloroplasts, in both bundle sheath and mesophyll cells. Levels of sucrose, glucose, and fructose were compared between wild-type and sxd1 plants. Mutant plants were fully capable of producing sucrose and accumulated all three sugars at concentrations above those measured in wild-type plants. Despite these increased sugar concentrations, photosynthetic gene expression was not significantly downregulated in affected areas of sxd1 leaf blades. These results are consistent with photosynthate being trapped within anthocyanin-accumulating regions of sxd1 leaves due to plasmodesmal occlusion at the bundle sheath-vascular parenchyma boundary of the minor veins. A model for SXD1 function is proposed in which the protein is involved in a chloroplast-to-nucleus signaling pathway necessary for proper late-stage differentiation of maize bundle sheath cells, including the developmentally regulated modification of plasmodesmata.

Amino Acid Sequence↗
PubMed1992

Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids.

Cell-to-cell spread of tobacco mosaic virus (TMV) is presumed to occur through plant intercellular connections, the plasmodesmata. Viral movement is an active process mediated by a specific virus-encoded P30 protein. P30 has at least two functions, to cooperatively bind single-stranded nucleic acids and to increase plasmodesmatal permeability. Here, we visualized P30 complexes with single-stranded DNA and RNA. These complexes are long, unfolded, and very thin (1.5 to 2.0 nm in diameter). Unlike TMV virions (300 x 18 nm), the complexes are compatible in size with the P30-induced increase in plasmodesmatal permeability (2.4 to 3.1 nm), making them likely candidates for the structures involved in the cell-to-cell movement of TMV. Mutational analysis using single and double deletion mutants of P30 revealed three regions potentially important for the protein function. Amino acid residues 65 to 86 possibly are required for correct folding of the active protein, and the regions between amino acid residues 112 to 185 and 185 to 268 potentially contain two independently active single-stranded nucleic acid binding domains designated binding domains A and B, respectively.

Amino Acid Sequence↗
PubMed1993

Alanine scanning mutagenesis of a plant virus movement protein identifies three functional domains.

Alanine scanning mutagenesis was performed on the red clover necrotic mosaic virus (RCNMV) movement protein (MP), and 12 mutants were assayed in vitro for RNA binding characteristics and in vivo for their ability to potentiate RCNMV cell-to-cell movement. The mutant phenotypes that were identified in vitro and in vivo suggest both that cooperative RNA binding is not necessary for cell-to-cell movement in vivo and that only a fraction of the wild-type RNA binding may be required. The MP mutants defined at least three distinct functional regions in the MP: an RNA binding domain, a cooperative RNA binding domain, and a third domain that is necessary for cell-to-cell movement in vivo. This third domain may be required for targeting the MP to cell walls and plasmodesmata, interacting with host proteins, folding, or possibly binding RNA into a functional ribonucleoprotein complex capable of cell-to-cell movement.

Amino Acid Sequence↗
PubMed1995

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.

Amino Acid Sequence↗
PubMed2004

Sequence of morphological and physiological events during natural ageing and senescence of a castor bean leaf: sieve tube occlusion and carbohydrate back-up precede chlorophyll degradation.

The development of castor bean (Ricinus communis L. var. sanguineus) leaves from bud break to abscission was studied to determine whether senescence of phloem precedes or follows chlorophyll degradation in the course of natural ageing of leaves. The castor bean leaf blade took 20 days for full expansion and its average life span was 60 days. From the day of full expansion on it suffered a substantial loss in N, a small loss in C, K and P and a gain in Ca, Mg and S. The content of soluble sugars increased with time, paralleled by a decrease of photosynthetic activity. Starch accumulated shortly before chlorophyll breakdown. The amino acid level in the leaves decreased steadily together with nitrate reductase and glutamine synthetase activity. Reactive oxygen species increased and oxidation-protecting compounds decreased during the life span of the leaves. Shortly after full leaf expansion an increasing number of sieve plates showed strong callose depositions when visualized by aniline blue method. At day 40 only half of the sieve tubes appeared functional. Chlorophyll breakdown followed these processes with a time lag of approximately 10 days. The sieve tube sap of ageing leaves had the same sucrose concentrations as young leaves, whereas amino acid concentrations decreased. High levels of reduced ascorbic acid and glutathione together with increasing levels of glutaredoxin indicated oxidative strain during senescence. We speculate that the gradual increase of reactive oxygen species during ageing together with the import of calcium ions lead to the stimulation of callose synthesis in plasmodesmata and sieve plates with the consequence of inhibition of phloem transport leading to carbohydrate back-up in the leaf blade. The latter may finally induce chlorophyll breakdown and, at the end, leaf abscission at the petiole base. Thus phloem blockage would precede and may be causal for chlorophyll degradation in leaf senescence.

Journal Article↗
PubMed2005

Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots.

Transgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36-67 kDa) under the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted to the endoplasmic reticulum (ER) remained inside the CCs and were used as 'zero trafficking' controls. In contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27-36 kDa, allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large proteins into the root tip. The presented data provide additional information on phloem development in Arabidopsis in relation to the formation of symplasmic domains.

Arabidopsis↗
PubMed2004

Nanotubular highways for intercellular organelle transport.

Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chemical synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small molecules. Accordingly, we propose a novel biological principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.

Actins↗
PubMed1995

Interaction of tobamovirus movement proteins with the plant cytoskeleton.

The movement protein of tobacco mosaic tobamovirus and related viruses is essential for the cell-to-cell spread of infection and, in part, determines the host range of the virus. Movement protein (MP) was fused with the jellyfish green fluorescent protein (GFP), and a modified virus that contained this MP:GFP fusion protein retained infectivity. In protoplasts and leaf tissues, the MP:GFP fusion protein was detected as long filaments shortly after infection. Double-labeling fluorescence microscopy suggests that the MP interacts and coaligns with microtubules. The distribution of the MP is disrupted by treatments that disrupt microtubules, but not by cytochalasin B, which disrupts filamentous F-actin. Microtubules may target the MP to plasmodesmata, the intercellular channels that connect adjacent cells.

Biological Transport↗
PubMed2000

Directed cell-to-cell movement of functional proteins: do transcription factors double as signal molecules in plants?

Cell fate in plants is predominantly determined by position during development. Jorgensen discusses a new mechanism by which cells can regulate gene expression in neighboring cells. Several lines of evidence suggest that transcription factors expressed in one cell can traffic through plasmodesmata to regulate gene expression in adjacent cells that are not expressing the transcription factor themselves. In plants, some transcription factors appear to be intercellular signaling molecules, as well as intracellular regulators of gene expression.

Cell Communication↗
PubMed1973

Morphogenesis and ultrastructure of Geotrichum candidum septa.

The ultrastructure and mode of formation of septa of Geotrichum candidum were investigated by light and electron microscopy. The invaginations of the lateral membrane and wall appear to initiate at multiple points around the circumference of the cell; the immature septum subsequently assumes a cart-wheel shape, with branched spokes radiating from the center of the septum. Each face of the septum is covered with a membrane possessing hitherto undescribed structural differentiation; the membrane substructures are comprised of two central subunits encircled by 12 identical subunits. The diameter of the entire 12 plus 2 structure is 24 to 25 nm, and the diameter of each individual subunit is approximately 4 nm. The maturation of the septum appears to occur by further deposition of material along the branched skeletal regions. Numerous small openings (micropores), formed as a result of incomplete deposition, ultimately give rise to plasmodesmata. During arthrospore formation, the plasmodesmal canals and associated micropores are occluded by electron-dense materials, rendering each segment of the hyphae completely independent of the rest of the hyphae.

Cell Membrane↗
PubMed2006

Disruption of microtubule organization and centrosome function by expression of tobacco mosaic virus movement protein.

The movement protein (MP) of Tobacco mosaic virus mediates the cell-to-cell transport of viral RNA through plasmodesmata, cytoplasmic cell wall channels for direct cell-to-cell communication between adjacent cells. Previous in vivo studies demonstrated that the RNA transport function of the protein correlates with its association with microtubules, although the exact role of microtubules in the movement process remains unknown. Since the binding of MP to microtubules is conserved in transfected mammalian cells, we took advantage of available mammalian cell biology reagents and tools to further address the interaction in flat-growing and transparent COS-7 cells. We demonstrate that neither actin, nor endoplasmic reticulum (ER), nor dynein motor complexes are involved in the apparent alignment of MP with microtubules. Together with results of in vitro coprecipitation experiments, these findings indicate that MP binds microtubules directly. Unlike microtubules associated with neuronal MAP2c, MP-associated microtubules are resistant to disruption by microtubule-disrupting agents or cold, suggesting that MP is a specialized microtubule binding protein that forms unusually stable complexes with microtubules. MP-associated microtubules accumulate ER membranes, which is consistent with a proposed role for MP in the recruitment of membranes in infected plant cells and may suggest that microtubules are involved in this process. The ability of MP to interfere with centrosomal gamma-tubulin is independent of microtubule association with MP, does not involve the removal of other tested centrosomal markers, and correlates with inhibition of centrosomal microtubule nucleation activity. These observations suggest that the function of MP in viral movement may involve interaction with the microtubule-nucleating machinery.

Animals↗